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Journal of Applied and Natural Science 6 (1): 290-293 (2014)
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Establishing monoxenic culture of arbuscular mycorrhizal fungus Glomus

intraradices through root organ culture
M. Srinivasan*, K. Kumar, K. Kumutha and P. Marimuthu
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore-641003 (Tamil Nadu),
INDIA
*Corresponding author. E-mail: srinimicrotech@gmail.com
Received: April 9, 2014; Revised received: May 5, 2014; Accepted: June 10, 2014

Abstract: Arbuscular mycorrhizal fungi are soil fungi distributed worldwide, forming symbiosis with most of the
vascular plants for their growth and survival, which is used for sustainable agriculture and ecosystem management.
This study investigated the establishment of monoxenic cultures of Glomus intraradices in association with
transformed carrot hairy root. The G.intraradices spores were isolated from sugarcane rhizosphere by wet sieving
and decanting technique and propagated in open pot culture. Transformation in to carrot hairy root was done using
Agrobacterium rhizogenes. Surface sterilization of G.intraradices spores co-cultured with transformed carrot hairy
root in Modified Strulla and Romand (MSR) medium was found the host root growth as well as for germination AM
spores. After three months of incubation in dark condition, significant production of extensive hyphal growth on
MSR medium and an average of 8500-9000 spores per petri dish was observed. The in vitro inoculum exhibited
higher potential of root colonization due to numerous intraradices mycelium with extensive spore load. The
produced monoxenic inoculum can be used in place of traditional system where it has a advantage of producing
contaminant free propagulas. Thus the monoxenic culture system, a powerful tool, of AM sporulation, can be used
for the mass production of monoxenic inoculum of AM fungi besides studying its biology.

Keywords: Carrot hairy root induction, Glomus intraradices, Monoxenic inoculum, MSR medium, Sugarcane
rhizosphere

INTRODUCTION first reported, the in vitro association of an Endogone

Continued increase in global population, with the
limitations in the world's supply of natural resources,
extensive use of chemical fertilizers and degeneration
of the environment is a major challenge to the agricul-
tural production today. Contrary to the chemical
fertilizers, organic manures and bioinoculants are less
expensive and achieving high productivity without
harming the environment. In order to implement such a
plan, the judicious use of nature's own biofertilizers
such as arbuscular mycorrhizal fungi (AMF) (Frank,
1885) are Obligate symbionts behavior belonging to
the phylum Glomeromycota (Schuessler et al., 2001)
that cannot complete their life cycle without establish-
ing a functional symbiosis with host plant. AMF
enhances the nutrient availability especially phospho-
rus, augment water uptake and induces resistant
against diseases and boost the crop yield (Lekberg and
Koids, 2005). Conventional methods available for
large scale production of AM fungi are pot cultivation
with sterilized soil, aeroponics, hydroponics and green
house based in vivo methods (Ijdo et al., 2011).
However these methods have limitation in high quality
inoculum production. Production of monoxenic AM
culture under in vitro conditions is one of the most
promising ways to obtain high number of spore
propagules in a shortest time with contamination
free inoculum (Binondo et al., 2012). Mosse (1962)
ISSN : 0974-9411 (Print), 2231-5209 (Online) All Rights Reserved © Applied and Natural Science Foundation www.ansfoundation.org
species with plant. In the mid-1970s, Mosse and
Hepper (1975) successfully established a culture of an
AM fungus associated with excised roots of tomato
(Lycopersicum esculentum Mill) and red clover
(T. pratense L.) in gelled medium. After this
primlinary work, ten years later the first observation of
in vitro AM inoculum production using carrot hairy
root was developed by Becard and Fortin in 1988.
Similarly Chabot et al. (1992) developed monoxenic
culture of AM using a mono-compartmental method.
Using another approach Declerck et al., 1996,
established in vitro AM inoculum by dual culture sys-
tem having sterilized mycorrhizal root segments and
Agrobacterium transformed carrot root. Fortin et al.
(2002) used split-plate method of monoxenic culture,
separating a proximal compartment containing the
carrot root and AM fungus from a distal compartment
to develop high density AM spore inoculum. Many
different strains of AM fungi have been developed in
monoxenic culture system. However, (Ijdo et al.,
2011) reported that only Glomus intraradices
species complex are fast colonizers that are able to
multiply around ten thousand in vitro propagules in
5-7 months of incubation. However, all the above
reported studies have taken a long period (around 7
monthes) to produce monoxenic AM spores using
different explants. This sporulation time when
291 M. Srinivasan et al. / J. Appl. & Nat. Sci. 6 (1): 290-293 (2014)

compare to conventional methods is much longer and

difficulty to commercialization. In order to produce
high quality inoculum with higher number of spores in
a relatively shorter period of time it is important to
develop suitable method for inoculum mass produc-
tion. Studies related to this area are less hence our
present study aims to mass produce high potential
monoxenic G. intraradices inoculum with help of
transformed carrot hairy root in shorter time span. Figs.1& 2. Hairy root induction from carrot after 1-2 week
incubation on the MS medium.
MATERIALS AND METHODS
Fungal inoculum propagation: G. intraradices fungi
used for this experiment was isolated from the
rhizosphere of sugarcane and multiplied in pot culture
of maize (Zea mays L. var-NK6240). The soil-sand
mix (2:1 w/w) substrates were sterilized in an
autoclave at 15 lb for half an hour to kill the
indigenones AMF propagules and to avoid cross
Fig. 3. Mass multiplication of Fig. 4. Co –Cultivation
contamination. After 60 of days incubation the soil transformed hairy root on the through Root organ culture.
samples were collected from pot, spores were isolated MSR medium.
by wet-sieving and decanting technique (Daniels and
Skipper, 1982). The mycorrhizal root were removed
from pot culture, estimation of AM colonization
was done by root clearing and staining technique
(Phillips and Hayman, 1970)
Sterilization of spores: The G. intraradices spores
were surface sterilized according to Becard and Piche
(1992) by showing then for 10 min in a 2 % w/v
Chloramine-T with Tween 20 (0.1 % v/v) which then Fig.5. In vitro G. intraradi- Fig.6. Extensive hyphal
ces spore germination and network growth.
followed by 30 min in antibiotic solution containg germ tube growth..
(Streptomycin 200 mg/lit, Ampicillin 200 mg/lit. Then
spores were rinsed for several times with sterile dis-
tilled water and store at 40C.
Carrot hairy root culture: Freshly harvested carrots
were surface sterilized using 0.1 % HgCl2 for 10 min
with continuous stirring. They were further rinsed
three times (each for 5 min) in sterile distilled water
and then dipped in 70 % ethanol for 30 sec and
superficially flamed and peeled out. Each carrot was Fig.7. Formation of new G. Fig. 8. In vitro mass produc-
sliced into 0.5 cm thick discs and were placed on 0.5 % intraradices spores from tion G. intraradices spores on
MS (Murashige and Skoog1962) plates with the basal germ tube. the MSR medium.
sides facing upwards. A loopful of 48 hours old
A. rhizogenes (MTCC-532) strain was pricked
manually for wounding on carrot surface and incubated
at 28ºC in dark for 2-3 weeks.
Monoxenic culture of AM inoculum production by
root organ culture (ROC): The surface sterilized G.
intraradices spores, and Ri-t-DNA transformed carrot
hairy root were associated routinely in Modified
Strulla and Romand (MSR) medium (Strulla and
Fig.9. Cluster of mature in Fig.10. Presence of internal
Romand 1986). The MSR mediun composed of (g/lit)
vitro G. intraradices hyphae and vesicles with in
MgSO47H2O – 73.9, KNO3-7.6, KCl -6.5, KH2PO4- spores. mycorrizal infected carrot
0.41 Ca(NO3)2.4 H2O-35.9, NaFeEDTA-0.16, root.
microelements-MnSO4.4H2O-1.225 CuSO4.5H20-1.1,
adding gallon gum (3g/lit) after that the medium was
ZnSO4.7H2O-0.14, H3BO3-0.925, Na2MOO4.-2H2O-
sterilized @1210C for 15 mins. 10-15 spores of AM
0.12 (NH4)6 Mo7O24.4H2O-1.7, Vitamins-Calcium
fungi were placed in different location near the emerg-
panthotenate-0.09, Biotin-0.0001, Nicotinic acid-0.1,
ing growth tip of pre grown carrot hairy root. Then
Pyridoxine-0.09, Thiamine-0.1 and Cyanocobalamine-
plates were incubated in inverted position at 27 oC in
0.04 Sucrose- 10. The pH was adjusted to 5.5 before
the dark condition.
 M. Srinivasan et al. / J. Appl. & Nat. Sci. 6 (1): 290-293 (2014)
RESULTS
Assessment of colonization potential of
G. intraradices in pot culture: The root colonization
potential of isolated G. intraradices AM fungus under
green house condition was 45 percent and the
maximum spore count (140 /100g of soil) was
observed in sand mixture substrate after 60 days of
inoculation. Plants subsequently grown without AMF
had no sign of AM colonization. The results clearly
indicated that the isolated G. intraradices spore is
alive and viable with ability to colonize roots and
multiply quickly at sixty days of inoculation.
Agrobacterium mediated- transformation: After 10
to 15 days of inoculation with Agrobacterium
rhizogenes, callus induction was observed on the
surface of carrot discs, followed by appearance of the
transformed roots on the side wall of discs (Fig. 1).
Hairy root initiation continued to occur upto 2-3
weeks (Fig. 2). A typical hairy root growth was
observed the appearance of numerous lateral roots
(Fig. 3) and its characteristic exhibiting the negative
geotrophic growth habit. These transformed hairy roots
were subsequently subcultured in fresh MS medium
containing antibiotic solution, cefotaxime at 250 mg/l
(HiMedia,Mumbai, India) to make it free from
A. rhizogenes. The bacterial free hairy roots (around 6
cm long) were cut, transferred to a MSR medium and
incubated for 5 days at 27 oC in the dark condition.
In vitro propagation G. intraradices: After seven
days of co-cultivation, (Fig.4) the spore germination
and hyphal growth was observed from surface
sterilized G. intraradices spores (Fig. 5). with
simultaneous growth of germ tube towards hairy root .
After the initial contact between the germ tube and the
root, the intercellular colonization took place on the
12th day . During 18-20 days of incubation germ tube
produced, multiply laterial branches on the root and
media surface by extensive hyphal proliferation
(Fig.6). After that, rapid enlargement of mycelium in
the form of clusters and the formatiom if new spores
(Fig.7) was observed within 30 days. The rate of spore
formation was slow during next 30 Days which was
followed by a rapid increase in spore number on 70th
days of incubation was observed. An average of 8500-
9000 spores per petri dish was produced after 3 months
of incubation (Figs. 8,9)
Three month old roots were harvested from prtidish, in
vitro AM colonization was observed by root
clearing and staining technique, the stained roots
showed numerous internal hyphae, arbuscules and
vesicles. The percentage of mycorrhizal colonization
around 75-80 % was recorded (Fig. 10)
DISCUSSION
Carrot is one of the most suitable and well known
model plant species for hairy root production (Bidondo
et al., 2012).In this present study, it was observed that
inoculation of carrot discs with 48 hours old
A. rhizogenes (MTCC-532) culture and incubated in
292
darkness at 27oC provided a suitable condition for
bacterial strains to insert their copies of Ri t-DNA.
These findings were pointed out by Mugnier and
Mosse, (1987) who observed that transformation
efficiency is highly dependent on the bacterial strain
used, which is related to the type of vir plasmid, binary
plasmid or bacterial chromosomal background as a
factor influencing hairy root initiation. The
co-cultivation of G. intraradices spores with carrot
hairy root was used in present study, because of their
comfort of propagation and better adaption in culture
than normal root. This result is supported by Douds
(2002) and Gadkar et al. (2006), where transformed
Daucus carota DC1 and DC2 hairy roots respectively
have been successfully used to initiate monoxenic
culture of AM fungi through root organ culture. In our
study 3-7 days Co- Cultivation with transformed carrot
root and G. intraradices spores, the hyphal growth was
moves towards host root. Desouza and Berbara, 1999
observed that 80 % of spore germination and hyphal
growth, on the 14th day of incubation. Hyphal growth
lengths of over 10 mm from germination spores have
been reported under the best experimental conditions
(Douds and schenck,1991) After that germ tube
branched and auxiliary cells grew in all directions
followed by hyphae proliferation numerously near the
root and density of hyphae on the surface of the
medium. The same result was noted by Costa et al.
(2013) who observed that in in vitro culture of
Gigaspora decipiens and Clomus clarum produced
typical structures like branched asorbing structure
(BAS) after spores germination. Pratap chandran and
potty (2010) also reported that in vitro co-culture of
AM fungi C. microcarpum with Vigna vexilate hairy
root on the 12-20 days of incubation 60 % of media
surface was covered with heavy mycelial network
growth and fan-like structures were observed. After 30
days of incubation, it was observed that in this study
heavy sporulation took place with extensive hyphal
network. We observed numerous vegetative spores of
G. intraradices were produced in the medium after two
months and spores matured to light brown colour to a
dark brown colour (Fig. 9). In our investigation, the
rate of sporulation was higher after 70 days
incubation. The reason might be due to the decrease of
sucrose and other mineral component level in the
medium. Similar trend was observed by James et al.
(2013) who showed that the rate of sporulation
depended on the sucrose concentration of the media.
Medium supplement with sucrose had less sporulation
than the medium without sucrose. This result was also
supported by the study of Diop (1994) and Clark
(1997) on Water agar medium without supplement
mineral also showing support to sporulation of AM
spores. In the present study MSR medium was most
appropriate medium, through which approximately
8500 to 9000 spores can be produced per plate after 3
months of root organ culture. Ijdo et al. (2011) also
reported that M medium (Becard and Fortin 1988) and
MSR medium (Strullu and Romand 1986) are
293 M. Srinivasan et al. / J. Appl. & Nat. Sci. 6 (1): 290-293 (2014)
frequently used for culture AM fungi on ROC.
Declarck et al. (1996) developed MSR medium to
optimize the growth of intracellular mycelium and
expensive sporulation of the fugues under in vitro
condition. In event of sporulation Declerck et al.
(2001) found same trends when carrot was used as host
plant to produce 8,400 Glomus intraradices spores per
petri plate after 12 weeks of incubation.
Conclusion
The root organ cultural method produced mycorrhizal
root segments holding G. intraradices spore with
Agrobacterium that transformed carrot hairy root as
host partner on MSR medium, establishing mass
production of pathogen free G. intraradices AM
inoculum. This ROC method providing extensive
monoexnic spore production in a small space and over
short period of time. This type of AM inoculum
production would significantly increase the number of
spore loads, to be inoculated in field, greatly
influencing the production of agricultural and
horticulture crops.
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